Fusaric acid induces DNA damage and post-translational modification Of p53 in hepatocellular carcinoma (HepG2) cells.

Master of Medical Science in Medical Biochemistry and Chemical Pathology. University of KwaZulu-Natal, Durban 2016.Abstract available in PDF file

Induction of Caspase-Mediated Apoptosis in HepG2 Liver Carcinoma Cells Using Mutagen–Antioxidant Conjugated Self-Assembled Novel Carbazole Nanoparticles and In Silico Modeling Studies

In this study, novel self-assembled carbazole-thiooctanoic acid nanoparticles (CTNs) were synthesized from amino carbazole (a mutagen) and thiooctanoic acid (an antioxidant). The nanoparticles were characterized using hyperspectral techniques. Then, the antiproliferative potential of CTNs was determined in HepG2 liver carcinoma cells. This study employed a solvent-antisolvent interaction method to synthesize a spherical CTN of size less than 50 nm. Moreover, CT was subsequently capped to gold nanoparticles (AuNPs) in the additional comparative studies. The CT derivative was synthesized from carbazole and lipoic acid by the amide bond formation reaction using a coupling agent. Furthermore, it was characterized using infrared (IR), 1H nuclear magnetic resonance, dynamic light scattering (DLS), and transmission electron microscopy techniques. The CT-capped gold nanoparticles (CTAuNPs) were prepared from CT, chloroauric acid, and NaBH4. The CTAuNPs were characterized using ultraviolet-visible, high-resolution TEM, DLS, and Fourier transform IR techniques. The cytotoxicity and apoptosis-inducing ability of both nanoparticles were determined in HepG2 cells. The results demonstrate that CTNs exhibit antiproliferative activity in the cancerous HepG2 cells. Moreover, molecular docking and molecular dynamics studies were conducted to explore the therapeutic potential of CT against human EGFR suppressor protein to gain more insights into the binding mode of the CT, which may show a significant role in anticancer therapy

Fumonisin B1 Epigenetically Regulates PTEN Expression and Modulates DNA Damage Checkpoint Regulation in HepG2 Liver Cells

Fumonisin B1 (FB1), a Fusarium-produced mycotoxin, is found in various foods and feeds. It is a well-known liver carcinogen in experimental animals; however, its role in genotoxicity is controversial. The current study investigated FB1-triggered changes in the epigenetic regulation of PTEN and determined its effect on DNA damage checkpoint regulation in human liver hepatoma G2 (HepG2) cells. Following treatment with FB1 (IC50: 200 µM; 24 h), the expression of miR-30c, KDM5B, PTEN, H3K4me3, PI3K, AKT, p-ser473-AKT, CHK1, and p-ser280-CHK1 was measured using qPCR and/or Western blot. H3K4me3 enrichment at the PTEN promoter region was assayed via a ChIP assay and DNA damage was determined using an ELISA. FB1 induced oxidative DNA damage. Total KDM5B expression was reduced, which subsequently increased the total H3K4me3 and the enrichment of H3K4me3 at PTEN promoters. Increased H3K4me3 induced an increase in PTEN transcript levels. However, miR-30c inhibited PTEN translation. Thus, PI3K/AKT signaling was activated, inhibiting CHK1 activity via phosphorylation of its serine 280 residue preventing the repair of damaged DNA. In conclusion, FB1 epigenetically modulates the PTEN/PI3K/AKT signaling cascade, preventing DNA damage checkpoint regulation, and induces significant DNA damage

Fusaric acid induces hepatic global m6A RNA methylation and differential expression of m6A regulatory genes in vivo - a pilot study

N6-methyladenosine (m6A) is an abundant epitranscriptomic mark that regulates gene expression to execute cellular developmental programmes and environmental adaptation. Fusaric acid (FA) is a mycotoxin that contaminates agricultural foods and exerts toxicity in humans and animals; however, its epitranscriptomic effects are unclear. We investigated the effect of FA on global m6A RNA methylation and mRNA expression levels of key m6A regulatory genes in C57BL/6 mouse livers. C57BL/6 mice (n = 6/group) were orally administered 0.1 M phosphate-buffered saline (PBS) or 50 mg/kg FA. Mice were euthanized 24 h after oral administration, livers were harvested, and RNA was isolated. RNA samples were assayed for global m6A levels using an m6A RNA Methylation Quantification Kit. The mRNA expression of m6A regulators i.e. writers, erasers, and readers were measured by qRT-PCR. FA increased global m6A RNA methylation (p < 0.0001) in mouse livers. FA increased the expression of METTL3 (p = 0.0143) and METTL14 (p = 0.0281), and decreased the expression of FTO (p = 0.0036) and ALKBH5 (p = 0.0035). The expression of YTHDF2 (p = 0.0007), YTHDF3 (p = 0.0061), and YTHDC2 (p = 0.0258) were increased by FA in mouse livers. This study shows that the liver m6A epitranscriptome can be modified by FA exposure in an in vivo model and can be useful for identifying the molecular mechanisms whereby m6A RNA modifications influence the toxicological outcomes of FA exposure

A Critical Review of the Biochemical Mechanisms and Epigenetic Modifications in HIV- and Antiretroviral-Induced Metabolic Syndrome

Metabolic syndrome (MetS) is a non-communicable disease characterised by a cluster of metabolic irregularities. Alarmingly, the prevalence of MetS in people living with Human Immunodeficiency Virus (HIV) and antiretroviral (ARV) usage is increasing rapidly. This study aimed to look at biochemical mechanisms and epigenetic modifications associated with HIV, ARVs, and MetS. More specifically, emphasis was placed on mitochondrial dysfunction, insulin resistance, inflammation, lipodystrophy, and dyslipidaemia. We found that mitochondrial dysfunction was the most common mechanism that induced metabolic complications. Our findings suggest that protease inhibitors (PIs) are more commonly implicated in MetS-related effects than other classes of ARVs. Furthermore, we highlight epigenetic studies linking HIV and ARV usage to MetS and stress the need for more studies, as the current literature remains limited despite the advancement in and popularity of epigenetics

<i>Moringa oleifera</i> Lam Leaf Extract Stimulates NRF2 and Attenuates ARV-Induced Toxicity in Human Liver Cells (HepG2)

The World Health Organization (WHO) reported that there are 37 million individuals living with the human immunodeficiency virus (HIV) worldwide, with the majority in South Africa. This chronic disease is managed by the effective use of antiretroviral (ARV) drugs. However, with prolonged use, ARV drug-induced toxicity remains a clinically complex problem. This study investigated the toxicity of ARV drugs on mitochondria and the NRF2 antioxidant pathway and its possible amelioration using Moringa oleifera Lam (MO) leaf extracts. This medicinal plant has a range of functional bioactive compounds. Liver (HepG2) cells were treated with individual ARV drugs: Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC), and Lamivudine (3TC) for 96 h, followed by MO leaf extracts for 24 h. Intracellular ROS, cytotoxicity, lipid peroxidation, total and reduced glutathione (GSH), ATP, and mitochondrial polarisation were determined. Finally, protein (pNRF2, NRF2, SOD2, CAT, and Sirt3) and mRNA (NRF2, CAT, NQO1 SOD2, Sirt3, and PGC1α) expression were measured using Western blot and qPCR, respectively. TDF, FTC, and 3TC significantly increased intracellular ROS and extracellular levels of both MDA and LDH. ARVs also reduced the GSH and ATP levels and altered the mitochondrial polarization. Further, ARVs reduced the expression of NRF2 SOD2, Sirt3, CAT, NQO1, UCP2 and PGC1α mRNA and consequently pNRF2, NRF2, SOD2, Sirt3 and CAT protein. In contrast, there was a significant reduction in the extracellular MDA and LDH levels post-MO treatment. MO significantly reduced intracellular ROS while significantly increasing GSH, ATP, and mitochondrial membrane polarization. The addition of MO to ARV-treated cells significantly upregulated the expression of NRF2, SOD2, Sirt3, CAT, UCP2, PGC1α, and NQO1 mRNA and pNRF2, NRF2, SOD2, Sirt3 proteins. Thus, MO ameliorates ARV-induced hepatotoxicity by scavenging oxidants by inducing the NRF2 antioxidant pathway. MO shows great therapeutic potential and may be considered a potential supplement to ameliorate ARV drug toxicity

Fusaric acid-induced promoter methylation of DNA methyltransferases triggers DNA hypomethylation in human hepatocellular carcinoma (HepG2) cells

Fusaric acid (FA), a mycotoxin contaminant of maize, displays toxicity in plants and animals; however, its epigenetic mechanism is unknown. DNA methylation, an epigenetic modification that regulates gene expression, is mediated by DNA methyltransferases (DNMTs; DNMT1, DNMT3A, and DNMT3B) and demethylases (MBD2). The expression of DNMTs and demethylases are regulated by promoter methylation, microRNAs (miR-29b) and post-translational modifications (ubiquitination). Alterations in these DNA methylation modifying enzymes affect DNA methylation patterns and offer novel mechanisms of FA toxicity. We determined the effect of FA on global DNA methylation as well as a mechanism of FA-induced changes in DNA methylation by transcriptional (promoter methylation), post-transcriptional (miR-29b) and post-translational (ubiquitination) regulation of DNMTs and MBD2 in the human hepatocellular carcinoma (HepG2) cell line. FA induced global DNA hypomethylation (p < 0.0001) in HepG2 cells. FA decreased the mRNA and protein expression of DNMT1 (p < 0.0001), DNMT3A (p < 0.0001), and DNMT3B (p < 0.0001) by upregulating miR-29b (p < 0.0001) and inducing promoter hypermethylation of DNMT1 (p < 0.0001) and DNMT3B (p < 0.0001). FA decreased the ubiquitination of DNMT1 (p = 0.0753), DNMT3A (p = 0.0008), and DNMT3B (p < 0.0001) by decreasing UHRF1 (p < 0.0001) and USP7 (p < 0.0001). FA also induced MBD2 promoter hypomethylation (p < 0.0001) and increased MBD2 expression (p < 0.0001). Together these results indicate that FA induces global DNA hypomethylation by altering DNMT promoter methylation, upregulating miR-29b, and increasing MBD2 in HepG2 cells